DURHAM, N.C.—Duke University researchers have come up with a way to use CRISPR/Cas 9 technology to quickly screen the
non-coding portion of the human genome to better understand and eventually better treat various diseases. The study outlining their findings appeared online
in Nature Biotechnology on April 3, 2017, and is titled
“CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.”

Now, that being said, let’s switch gears rather abruptly to talk about the universe. Scientists have long been confused
by the amount of observable matter and energy in the universe and how many things don’t add up, leading to the dark energy and dark matter hypotheses
that state that a little under 70 percent of the universe is dark energy and a little over a quarter of it is dark matter—all “normal”
matter and energy observable by current instruments is, theoretically, only 5 percent of the universe or less.

The
connection between the genome and “dark matter” is in the non-coding DNA—often called “junk DNA” in the past. What was long
thought to be extraneous genetic matter actually turns out to be functional, and thus this largely unknown and long-ignored part of our genetics is now often
colloquially referred to as the dark matter of the genome.

And now, back to Duke University and its researchers
and their own journey into the dark places, which they believe could revolutionize understanding of the genetically inherited risks of developing heart
disease, diabetes, cancer, neurological disorders and others—thus leading to new and better treatments.

“Identifying single mutations that cause rare, devastating diseases like muscular dystrophy has become relatively straightforward,” said
Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke University. “But more common diseases that run in families
often involve lots of genes as well as genetic reactions to environmental factors. It’s a much more complicated story, and we’ve been wanting a
way to better understand it. Now we’ve found a way.”

The new technique created at Duke uses as its
foundation the “gene-hacking” system known as CRISPR/Cas9. As Duke explains, it was originally discovered as a natural antiviral defense
mechanism in bacteria, noting, “the system recognizes and homes in on the genetic code of previous intruders and then chops up their DNA.”

And, of course, in recent years, researchers have learned to use this very system to do precise “cut and
paste” operations in DNA sequences in living organisms, opening up advances in creating animal models, potentially finding new gene therapy approaches
and more.

In the current study at Duke, researchers added molecular machinery that can control gene activity by
manipulating the web of biomolecules that determines which genes each cell activates and to what degree. And, getting back to that earlier cosmological
segue, Duke explains that with the new CRISPR/Cas 9 tool, Gersbach and his colleagues are exploring the 98 percent of our genetic code that is the dark
matter of the genome.

“Only a small fraction of our genome encodes instructions to make proteins that guide
cellular activity,” explained Tyler Klann, the biomedical engineering graduate student who led the work in Gersbach’s lab. “But more than
90 percent of the genetic variation in the human population that is associated with common disease falls outside of those genes. We set out to develop a
technology to map this part of the genome and understand what it is doing.”

The answer, says Klann, lies
with promoters and enhancers. Promoters sit directly next to the genes they control. Enhancers, however, which modulate promoters, can be just about anywhere
due to the genome’s complex 3D geometry, making it difficult to discern what they are actually doing.

“If an enhancer is dialing a promoter up or down by 10 or 20 percent, that could logically explain a small genetic contribution to
cardiovascular disease, for example,” said Gersbach. “With this CRISPR-based system, we can more strongly turn these enhancers on and off to see
exactly what effect they’re having on the cell. By developing therapies that more dramatically affect these targets in the right direction, we could
have a significant effect on the corresponding disease.”

As Duke points out in talking about Gerbach’s
work, “that’s all well and good for exploring the regions of the genome that researchers have already identified as being linked to diseases, but
there are potentially millions of sites in the genome with unknown functions” and that, Duke notes, meant diving down into “the dark genome
rabbit hole.”

But Gersbach and his team didn’t dive down it alone, instead turning to colleagues Greg
Crawford, an associate professor of pediatrics and medical genetics, and Tim Reddy, an assistant professor of bioinformatics and biostatistics. All three
professors work together in the Duke Center for Genomic and Computational Biology.

Crawford developed a way of
determining which sections of DNA are “open for business.” That is, Duke notes, which sections are not tightly packed away, providing access for
interactions with biomachinery such as RNA and proteins. These sites, the researchers reason, are the most likely to be contributing to a cell’s
activity in some way. Reddy has been developing computational tools for interpreting these large genomic data sets.

Over the past decade, Crawford has scanned hundreds of types of cells and tissues affected by various diseases and drugs and come up with a list of
more than two million potentially important sites in the dark genome.

Investigating so many sites one at a time
is, clearly, not feasible. In the new study, Crawford, Reddy and Gersbach demonstrate a high-throughput screening method to investigate many of these
potentially important genetic sequences in short order. The initial studies screened hundreds of these sites across millions of base pairs of the genome.
This is only a start, of course, and the researchers are already working to scale this up by a hundredfold or even ten times that.

“Small molecules can target proteins and RNA interference targets RNA, but we needed something to go in and modulate
the non-coding part of the genome,” said Crawford. “Up until now, we didn’t have that.”

The technique is already producing results, identifying previously known genetic regulatory elements while also spotting a few new ones. The results
also showed it can be used to turn genes either on or off, which is superior to other tools for studying biology which only turn genes off. Different cell
types also produced different—but partially overlapping—results, highlighting the biological complexity in gene regulation and disease that can
be interrogated with this technology.

“Now that we have this tool, we can go in and annotate the functions
of these previously unknown but important stretches of our genome,” commented Gersbach. “With so many places to look, and the ability to do it
quickly and robustly, we’ll undoubtedly find new segments that are important for disease, which will provide new avenues for developing
therapeutics.”